Involvement of different calcium channels in K+- and veratridine-induced increases of cytosolic calcium concentration in rat cerebral cortical synaptosomes.
Intracellular calcium ion concentrations ([Ca2+]i) in rat cerebral cortical synaptosomes were measured, using the calcium chelating fluorescence dye fura-2. The synaptosomes were depolarized by elevation of the extracellular K+ concentration or by addition of veratridine, which opens voltage-dependent Na+-channels and prevents their inactivation. Both enhancement of the concentration of extracellular K+ (up to 60 mM) and veratridine (1-100 microM) increased the [Ca2+]i in a concentration-dependent manner. In the absence of extracellular Ca2+, the K+- and veratridine-induced increases in [Ca2+]i were abolished, indicating that the increase in [Ca2+]i was due to an influx of extracellular Ca2+. Tetrodotoxin (TTX), a blocker of the voltage-dependent Na+ channel, inhibited the veratridine-induced (10 microM) Ca2+ influx by more than 80%, while the K+-evoked (30 mM) increase of [Ca2+]i was TTX-resistant. Both the K+- and the veratridine-induced Ca2+ influx were not reduced by nifedipine (1 microM), a blocker of L-type Ca2+ channels. Blockade of the voltage dependent N-type Ca2+ channels with omega-conotoxin GVIA (omega-CTx GVIA; 0.1 microM) and of the voltage-dependent P/Q-type channels with omega-agatoxin IVA (omega-AgaTx IVA; 0.2 microM) inhibited the K+-induced increase in [Ca2+]i by about 30 and 55%, respectively; these effects were additive. Omega-conotoxin MVIIC (omega-CTx MVIIC) at a concentration of 0.2 microM, which may be assumed to block predominantly the Q-type Ca2+ channel, inhibited the K+-induced increase in [Ca2+]i by 50%. The veratridine-induced increase in [Ca2+]i was reduced by about 25% by omega-CTx GVIA (0.1 microM), but was resistant to omega-AgaTx IVA (0.2 microM) and omega-CTx MVIIC (0.2 omegaM). Mibefradil (former designation Ro 40-5967), a Ca2+ antagonist which blocks all types of voltage-dependent Ca2+ channels including the T and R channels, led to a concentration-dependent inhibition of the K+- and veratridine-induced increase in [Ca2+]i (abolition at 10 microM mibefradil). Ifenprodil, another non-specific blocker of voltage-dependent Ca2+ channels, also inhibited the K+- and veratridine-induced increase in [Ca2+]i in concentration-dependent manner and abolished it at 320 microM ifenprodil. In contrast, KB-R 7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; 1 and 3 microM), a highly potent and selective inhibitor of the Na+/Ca2+ exchanger (NCX1), failed to inhibit the K+- and veratridine-induced increase in [Ca2+]i. It is concluded that the K+-induced increase in free cytosolic Ca2+ results from Ca2+ influx through voltage-dependent N- and, above all, Q-type Ca2+ channels. N-type Ca2+ channels also play a minor role in the veratridine-induced increase in [Ca2+]i, but P/Q-type channels do not appear to be involved at all. The inhibition of the veratridine-induced, omega-CTx GVIA- and omega-AgaTx IVA-resistant increase in [Ca2+]i by mibefradil and the failure of KB-R 7943 to inhibit this response are compatible with the suggestion that in rat cerebral cortical synaptosomes, Ca2+ influx via the R-type Ca2+ channel and/or another so far uncharacterized Ca2+ channel may substantially contribute to the veratridine-induced increase in [Ca2+]i.
Meder
W
W
Institut für Pharmakologie und Toxikologie der Rheinischen Friedrich-Wilhelms-Universität Bonn, Germany.
Fink
K
K
Göthert
M
M
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology*; D002540:Cerebral Cortex / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D011188:Potassium / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D013574:Synaptosomes / Q000187:drug effects* / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology; D014701:Veratridine / Q000494:pharmacology*
C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D014701:Veratridine; D000251:Adenosine Triphosphatases; C034688:sodium calcium ATPase; D013890:Thiourea; D011188:Potassium; D002118:Calcium